Abstract
BackgroundGlycerol, a byproduct of biodiesel, has become a readily available and inexpensive carbon source for the production of high-value products. However, the main drawback of glycerol utilization is the low consumption rate and shortage of NADPH formation, which may limit the production of NADPH-requiring products. To overcome these problems, we constructed a carbon catabolite repression-negative ΔptsGglpK* mutant by both blocking a key glucose PTS transporter and enhancing the glycerol conversion. The mutant can recover normal growth by co-utilization of glycerol and glucose after loss of glucose PTS transporter. To reveal the metabolic potential of the ΔptsGglpK* mutant, this study examined the flux distributions and regulation of the co-metabolism of glycerol and glucose in the mutant.ResultsBy labeling experiments using [1,3-13C]glycerol and [1-13C]glucose, 13C metabolic flux analysis was employed to decipher the metabolisms of both the wild-type strain and the ΔptsGglpK* mutant in chemostat cultures. When cells were maintained at a low dilution rate (0.1 h−1), the two strains showed similar fluxome profiles. When the dilution rate was increased, both strains upgraded their pentose phosphate pathway, glycolysis and anaplerotic reactions, while the ΔptsGglpK* mutant was able to catabolize much more glycerol than glucose (more than tenfold higher). Compared with the wild-type strain, the mutant repressed its flux through the TCA cycle, resulting in higher acetate overflow. The regulation of fluxomes was consistent with transcriptional profiling of several key genes relevant to the TCA cycle and transhydrogenase, namely gltA, icdA, sdhA and pntA. In addition, cofactor fluxes and their pool sizes were determined. The ΔptsGglpK* mutant affected the redox NADPH/NADH state and reduced the ATP level. Redox signaling activated the ArcA regulatory system, which was responsible for TCA cycle repression.ConclusionsThis work employs both 13C-MFA and transcription/metabolite analysis for quantitative investigation of the co-metabolism of glycerol and glucose in the ΔptsGglpK* mutant. The ArcA regulatory system dominates the control of flux redistribution. The ΔptsGglpK* mutant can be used as a platform for microbial cell factories for the production of biofuels and biochemicals, since most of fuel molecule (e.g., alcohols) synthesis requires excess reducing equivalents.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0591-1) contains supplementary material, which is available to authorized users.
Highlights
Glycerol, a byproduct of biodiesel, has become a readily available and inexpensive carbon source for the production of high-value products
This study investigated the co-metabolism of glycerol and glucose in E. coli, and elucidated the metabolic potential of the ΔptsGglpK* mutant as a chassis for biosynthesis from cheap feedstock. 13C metabolic flux analysis (13C-MFA) provided rigorous comparison of cell fluxomes between the wild-type and the mutant strain using well-controlled chemostat cultures: (1) various growth rates could be realized by changing the dilution rate; and (2) cell metabolism could be maintained in a metabolic steady-state during labeling experiment [27, 28]
The wild-type strain consumed glucose and glycerol sequentially owing to carbon catabolite repression (CCR) (Fig. 1a), while the ΔptsGglpK* mutant co-metabolized both glucose and glycerol with faster glycerol consumption than glucose consumption (Fig. 1b; Additional file 1a)
Summary
A byproduct of biodiesel, has become a readily available and inexpensive carbon source for the production of high-value products. The main drawback of glycerol utilization is the low consumption rate and shortage of NADPH formation, which may limit the production of NADPH-requiring products. To overcome these problems, we constructed a carbon catabolite repression-negative ΔptsGglpK* mutant by both blocking a key glucose PTS transporter and enhancing the glycerol conversion. Genetic modification of the glpK gene has resulted in the change in this enzyme that is insensitive to FBP and EIIAGlc, allowing improved the glycerol consumption rate [10, 11] Another obstacle to glycerol utilization is the shortage of NADPH formation because minimal glycolytic flux is reverted from GAP upwards to the oxidative pentose phosphate (PP) pathway. Glucose utilization prevents the metabolism of glycerol because of carbon catabolite repression (CCR) [15]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
